Development of adeno-associated viral vectors for human gene therapy has been pursued since AAV appears to stably integrate into a unique locus on the human chromosome 19, is not associated with any known human disease, and appears to be capable of high levels of gene expression in non-dividing cells. Rep proteins encoded by the p5 promoter of AAV play an important role in these processes. Rep 68 and 79 are thought to be involved in several activities related to DNA replication as well as targeted integration. Characterization of the molecular events catalyzed by these proteins has ben impaired by the low abundance of the Rep proteins available for biochemical characterization. To obtain larger quantities of the Rep proteins, the AAV ORF was cloned and Rep expressed as a Maltose binding protein-Rep fusion protein in E. coli. This has led to the purification of 50-100 mg of homogeneous Rep 68 and Rep 78 which have the following activities indistinguishable from that expressed by wild-type Rep protein. These include 1) binding to the AAV inverted terminal repeats, 2) site-specific endonuclease activity at the terminal resolution site (trs) and DNA helicase activity. In addition we have initiated crystallographic studies of Rep proteins complexed with their specific DNA binding sequences. The over expressed Rep fusion proteins are also being utilized to detect interactions with host nuclear proteins that may regulate gene expression from the integrated recombinant AAV vectors.